scholarly journals Photoactivation of Cell-Free Expressed Archaerhodopsin-3 in a Model Cell Membrane

2021 ◽  
Vol 22 (21) ◽  
pp. 11981
Author(s):  
Navid Khangholi ◽  
Marc Finkler ◽  
Ralf Seemann ◽  
Albrecht Ott ◽  
Jean-Baptiste Fleury
Keyword(s):  
Plasma Membranes ◽  
Fluorescent Protein ◽  
Transmembrane Protein ◽  
Expression System ◽  
Light Sensitivity ◽  
Green Laser ◽  
Receptor Proteins ◽  
Electrophysiological Properties ◽  
Photoelectrical Properties

Transmembrane receptor proteins are located in the plasma membranes of biological cells where they exert important functions. Archaerhodopsin (Arch) proteins belong to a class of transmembrane receptor proteins called photoreceptors that react to light. Although the light sensitivity of proteins has been intensely investigated in recent decades, the electrophysiological properties of pore-forming Archaerhodopsin (Arch), as studied in vitro, have remained largely unknown. Here, we formed unsupported bilayers between two channels of a microfluidic chip which enabled the simultaneous optical and electrical assessment of the bilayer in real time. Using a cell-free expression system, we recombinantly produced a GFP (green fluorescent protein) labelled as a variant of Arch-3. The label enabled us to follow the synthesis of Arch-3 and its incorporation into the bilayer by fluorescence microscopy when excited by blue light. Applying a green laser for excitation, we studied the electrophysiological properties of Arch-3 in the bilayer. The current signal obtained during excitation revealed distinct steps upwards and downwards, which we interpreted as the opening or closing of Arch-3 pores. From these steps, we estimated the pore radius to be 0.3 nm. In the cell-free extract, proteins can be modified simply by changing the DNA. In the future, this will enable us to study the photoelectrical properties of modified transmembrane protein constructs with ease. Our work, thus, represents a first step in studying signaling cascades in conjunction with coupled receptor proteins.

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.

Activity of Cardiorespiratory Networks Revealed by Transsynaptic Virus Expressing GFP

2001 ◽  
Vol 85 (1) ◽  
pp. 435-438 ◽  
Author(s):  
Mustapha Irnaten ◽  
Robert A. Neff ◽  
Jijiang Wang ◽  
Arthur D. Loewy ◽  
Thomas C. Mettenleiter ◽  
...  
Keyword(s):  
Pseudorabies Virus ◽  
Fluorescent Protein ◽  
Nucleus Ambiguus ◽  
Dependent Manner ◽  
Neural Basis ◽  
Electrophysiological Properties ◽  
Functional Studies ◽  
Neuronal Systems ◽  
Green Fluorescent

A fluorescent transneuronal marker capable of labeling individual neurons in a central network while maintaining their normal physiology would permit functional studies of neurons within entire networks responsible for complex behaviors such as cardiorespiratory reflexes. The Bartha strain of pseudorabies virus (PRV), an attenuated swine alphaherpesvirus, can be used as a transsynaptic marker of neural circuits. Bartha PRV invades neuronal networks in the CNS through peripherally projecting axons, replicates in these parent neurons, and then travels transsynaptically to continue labeling the second- and higher-order neurons in a time-dependent manner. A Bartha PRV mutant that expresses green fluorescent protein (GFP) was used to visualize and record from neurons that determine the vagal motor outflow to the heart. Here we show that Bartha PRV-GFP-labeled neurons retain their normal electrophysiological properties and that the labeled baroreflex pathways that control heart rate are unaltered by the virus. This novel transynaptic virus permits in vitro studies of identified neurons within functionally defined neuronal systems including networks that mediate cardiovascular and respiratory function and interactions. We also demonstrate superior laryngeal motorneurons fire spontaneously and synapse on cardiac vagal neurons in the nucleus ambiguus. This cardiorespiratory pathway provides a neural basis of respiratory sinus arrhythmias.

scholarly journals Rotavirus-Like Particles: A Novel Nanocarrier for the Gut

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Naima G. Cortes-Perez ◽  
Catherine Sapin ◽  
Loïc Jaffrelo ◽  
Sabine Daou ◽  
Jean Pierre Grill ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Expression System ◽  
Bioactive Molecules ◽  
Intestinal Cells ◽  
Epithelial Cell Line ◽  
Intragastric Administration ◽  
Trinitrobenzene Sulfonic Acid ◽  
Reporter Protein

The delivery of bioactive molecules directly to damaged tissues represents a technological challenge. We propose here a new system based on virus-like particles (VLP) from rotavirus, with a marked tropism for the gut to deliver bio-active molecules to intestinal cells. For this, nonreplicative VLP nanoparticles were constructed using a baculovirus expression system and used to deliver an exogenous biomolecule, the green fluorescent protein (GFP), into either MA104 cells or intestinal cells from healthy and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-treated mice. Our results show that expression of rotavirus capsid proteins in baculovirus led to the auto assembly of VLP that display similar properties to rotavirus. In vitro experiments showed that VLP were able to enter into MA104 cells and deliver the reporter protein. Intragastric administration of fluorescent VLP in healthy and TNBS-treated mice resulted in the detection of GFP and viral proteins in intestinal samples. Our results demonstrate an efficient entry of non-replicative rotavirus VLP into the epithelial cell line MA104 and provide the first in vivo evidence of the potential of these nanoparticles as a promising safe candidate for drug delivery to intestinal cells.

scholarly journals Using Fluorescence Quenching Titration to Determine the Orientation of a Model Transmembrane Protein in Mimic Membranes

Materials ◽  
2019 ◽  
Vol 12 (3) ◽  
pp. 349 ◽  
Author(s):  
Haihong Huang ◽  
Baosheng Ge ◽  
Shuai Zhang ◽  
Jiqiang Li ◽  
Chenghao Sun ◽  
...  
Keyword(s):  
Ionic Strength ◽  
Fluorescence Quenching ◽  
Plasma Membranes ◽  
Ph Value ◽  
Biological Activities ◽  
Transmembrane Protein ◽  
Membrane Curvature ◽  
Minor Effect ◽  
Fluorescence Quenching Titration

After synthesis of transmembrane proteins (TMPs), they are transferred and inserted into plasma membranes to play biological functions. Crucially, orientation of TMPs in membranes determines whether they have biological activities. In cellular environments, a number of cofactors, such as translocon, can assist TMPs to be inserted into membranes in defined orientations. During in vitro reconstitution of TMPs with mimic membranes, both insertion and orientation of TMPs are primarily determined by interactions with the membrane. Yet the knowledge is limited, hindering the in vitro applications of TMPs. Here, we take Bacteriorhodopsin (bR) as a model TMP, using fluorescence quenching titration experiment to identify orientation of bR in mimic membranes, examining effects of a number of factors, including lipid composition, pH value, ionic strength and membrane curvature. The most effective determinant is the lipid type, which modulates insertion and orientation of bR in membranes by changing the membrane surface charge and the membrane fluidity. Both the pH value and the ionic strength play secondary roles by tuning the nature of the electrostatic interaction. The membrane curvature was found to have a minor effect on orientation of bR in membranes. By comparing orientations of bR in folded and unfolded states, no obvious change was observed, informing that nascent proteins could be inserted into membranes in defined orientations before folding into the native state inside the membrane.

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 Synthetic promoter based azide biosensor toolkit to advance chemical-biology

2020 ◽  
Author(s):  
Chandra Kanth Bandi ◽  
Kyle S. Skalenko ◽  
Ayushi Agrawal ◽  
Neelan Sivaneri ◽  
Margaux Thiry ◽  
...  
Keyword(s):  
Chemical Biology ◽  
Fluorescent Protein ◽  
Low Cost ◽  
Expression System ◽  
Detection Methods ◽  
Synthetic Promoter ◽  
Bioorthogonal Reaction ◽  
Green Fluorescent

AbstractReal-time azide or azido-functionalized molecular detection inside living cells using bioorthogonal chemistry-based approaches has been revolutionary to advancing chemical-biology. These methods have enabled diverse applications ranging from understanding the role of cellular glycosylation pathways, identifying diseased cells, and targeting delivery of azido-based therapeutic drugs. However, while classical techniques were applicable only to in-vitro detection of such functional groups, even recent bioorthogonal based-detection methods require expensive sensing reagents and also cannot selectively identify inorganic azide. Here, we report an in-vivo synthetic promoter based azide biosensor toolkit to selectively detect azide anions. A promiscuous cyanate-specific promoter was engineered to detect azide and rapidly induce expression of green fluorescent protein (GFP) in Escherichia coli. Our synthetic azide operon allows highly-tunable GFP expression, outperforming the classic lac-operon, and also offers an alternative low-cost protein expression system. Finally, we showcase the utility of this toolkit for in-vivo bioorthogonal reaction biosensing and glycoengineering based applications.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Engineering of Recombinant Sheep Pox Viruses Expressing Foreign Antigens

2021 ◽  
Vol 9 (5) ◽  
pp. 1005
Author(s):  
Olga Chervyakova ◽  
Elmira Tailakova ◽  
Nurlan Kozhabergenov ◽  
Sandugash Sadikaliyeva ◽  
Kulyaisan Sultankulova ◽  
...  
Keyword(s):  
Fluorescent Protein ◽  
Viral Vector ◽  
Foot And Mouth Disease ◽  
Open Reading Frames ◽  
Foreign Gene ◽  
Mouth Disease Virus ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
Reading Frames

Capripoxviruses with a host range limited to ruminants have the great potential to be used as vaccine vectors. The aim of this work was to evaluate attenuated sheep pox virus (SPPV) vaccine strain NISKHI as a vector expressing several genes. Open reading frames SPPV020 (ribonucleotide kinase) and SPPV066 (thymidine kinase) were selected as sites for the insertion of foreign genes. Two integration plasmids with expression cassette were designed and constructed. Recombinant SPPVs expressing an enhanced green fluorescent protein (EGFP) (rSPPV(RRΔ)EGFP and rSPPV(TKΔ)EGFP), Foot-and-mouth disease virus capsid protein (VP1), and Brucella spp. outer membrane protein 25 (OMP25) (rSPPV(RRΔ)VP1A-(TKΔ)OMP25) were generated under the transient dominant selection method. The insertion of foreign genes into the SPPV020 and SPPV066 open reading frames did not influence the replication of the recombinant viruses in the cells. Successful foreign gene expression in vitro was assessed by luminescent microscopy (EGFP) and Western blot (VP1 and OMP25). Our results have shown that foreign genes were expressed by rSPPV both in permissive (lamb testicles) and non-permissive (bovine kidney, saiga kidney, porcine kidney) cells. Mice immunized with rSPPV(RRΔ)VP1A-(TKΔ)OMP25 elicited specific antibodies to both SPPV and foreign genes VP1 and OMP25. Thus, SPPV NISKHI may be used as a potential safe immunogenic viral vector for the development of polyvalent vaccines.

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