Inducible Expression of Transmembrane Proteins on Bacterial Magnetic Particles in Magnetospirillum magneticum AMB-1

ABSTRACT Bacterial magnetic particles (BacMPs) produced by the magnetotactic bacterium Magnetospirillum magneticum AMB-1 are used for a variety of biomedical applications. In particular, the lipid bilayer surrounding BacMPs has been reported to be amenable to the insertion of recombinant transmembrane proteins; however, the display of transmembrane proteins in BacMP membranes remains a technical challenge due to the cytotoxic effects of the proteins when they are overexpressed in bacterial cells. In this study, a tetracycline-inducible expression system was developed to display transmembrane proteins on BacMPs. The expression and localization of the target proteins were confirmed using luciferase and green fluorescent protein as reporter proteins. Gene expression was suppressed in the absence of anhydrotetracycline, and the level of protein expression could be controlled by modulating the concentration of the inducer molecule. This system was implemented to obtain the expression of the tetraspanin CD81. The truncated form of CD81 including the ligand binding site was successfully displayed at the surface of BacMPs by using Mms13 as an anchor protein and was shown to bind the hepatitis C virus envelope protein E2. These results suggest that the tetracycline-inducible expression system described here will be a useful tool for the expression and display of transmembrane proteins in the membranes of BacMPs.

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336 QUANTITATIVE ANALYSIS OF TETRACYCLINE-INDUCIBLE EXPRESSION OF THE GREEN FLUORESCENT PROTEIN GENE IN TRANSGENIC CHICKENS

Reproduction Fertility and Development ◽

10.1071/rdv25n1ab336 ◽

2013 ◽

Vol 25 (1) ◽

pp. 315

Author(s):

B. Koo ◽

M. Kwon ◽

J. Roh ◽

J. Kim ◽

T. Kim

Keyword(s):

Fluorescent Protein ◽

Expression System ◽

Constitutive Expression ◽

Transgenic Animals ◽

Inducible Expression ◽

Gfp Expression ◽

Transgenic Chicken ◽

Inducible Expression System ◽

Green Fluorescent ◽

Transgenic Chickens

The use of transgenic farm animals as bioreactors to address the growing demand for biopharmaceuticals, both in terms of increased quantity and greater number, represents a key development in the advancement of medical science. However, the potential for detrimental side effects as a result of uncontrolled constitutive expression of foreign genes in transgenic animals is a well-recognised limitation of such systems. Previously, using a tetracycline-inducible expression system, we demonstrated the induction of expression of a transgene encoding green fluorescent protein (GFP) in transgenic chickens by feeding with doxycycline, a tetracycline derivative; expression of GFP reverted to pre-induction levels when the inducer was removed from the diet (Kwon et al. 2011 Biochem. Biophys. Res. Commun. 410, 890–894). As a proof of principle study, however, quantitative assessment of expression was not possible, as only 1 G0 and 1 G1 transgenic chicken was obtained. In the current study, with 7 G2 transgenic chickens obtained from 1 G1 hen, we confirmed stable genomic integration of a single copy number of the transgene by Southern blot analysis. As we have observed in G1 transgenic chicken previously, all of the G2 transgenic chickens emitted a green fluorescence upon doxycycline feeding (50 mg kg–1 of formula feed). Fluorescence became detectable 4 days after starting doxycycline feeding, and maximum GFP expression was detected after 2 weeks. Removal of doxycycline from the diet after 14 days of induction feeding resulted in the return of external fluorescence to pre-induction levels after 39 days. Quantitative analysis of gene induction was done using protein and mRNA extracted from primary cultured cells derived from 6-day transgenic chicken embryos. The eggs were obtained by mating a nontransgenic wild-type hen with 1 of G2 transgenic roosters. Protein levels of GFP were analysed by immunoblot and quantified using a densitometer. In the absence of doxycycline, the amount of GFP per 1 µg of total protein was 0.2 ng. However, when the cells were treated with doxycycline for 6 days, the amount of GFP increased to 3.1 ng per 1 µg of total protein, which was 16-fold higher than that of the cells pre-treated with doxycycline. Switching to doxycycline-free medium after doxycycline induction resulted in significant abrogation of GFP expression in 6 days; the amount of GFP reduced from 3.1 to 0.5 ng, a 6.2-fold reduction. Transcription of the GFP gene was also assessed by Northern blot. The amount of GFP mRNA measured by band density increased as much as 20-fold (3.9/0.2) with 6 days of doxycycline induction and declined to 1/8 (3.9/0.5) when doxycycline was removed from the cell culture media for 6 days. The use of an inducible expression system that can be regulated by dietary supplementation could help mitigate the physiological disruption that can occur in transgenic animals as a result of uncontrolled constitutive expression of a transgene.

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Site-selective immobilization of streptavidin on enzymatically biotinylated bacterial magnetic particles

MRS Proceedings ◽

10.1557/proc-1094-dd07-19 ◽

2008 ◽

Vol 1094 ◽

Author(s):

Yoshiaki Maeda ◽

Tomoko Yoshino ◽

Haruko Takeyama ◽

Masaaki Takahashi ◽

Harumi Ginya ◽

...

Keyword(s):

Magnetic Nanoparticles ◽

Magnetic Particles ◽

Fluorescent Protein ◽

Functional Materials ◽

Fluorescence Analysis ◽

Bacterial Cells ◽

Conjugation System ◽

Bacterial Magnetic Particles ◽

Site Selective

AbstractBiotinylated magnetic nanoparticles were constructed by displaying biotin acceptor peptide (BAP) on the surfaces of bacterial magnetic particles (BacMPs) synthesized by Magnetospirillum magneticum AMB-1. Both BAP and green fluorescent protein (GFP) were fused to Mms13 that was isolated from BacMP membranes. The localization of the fusion protein, BAP-Mms13-GFP, was confirmed by fluorescence analysis. BacMPs that expressed BAP-Mms13-GFP (BAP/GFP-BacMPs) were extracted from bacterial cells and incubated with biotin and Escherichia coli biotin ligase. The in vitro biotinylation of BAP/GFP-BacMPs was confirmed using alkaline phosphatase (ALP)-streptavidin detection. The conjugation system developed in this study provides a method for producing biotin- or streptavidin-labeled magnetic nanoparticles without the use of a crosslinker reagent. Various functional materials can be immobilized site-selectively onto these uniquely designed BacMPs. By combining this site-selective biotinylation technology and protein display methodology, increasingly innovative and attractive magnetic nano-materials can be constructed.

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Development of a Tet-On Inducible Expression System for the Anhydrobiotic Cell Line, Pv11

Insects ◽

10.3390/insects11110781 ◽

2020 ◽

Vol 11 (11) ◽

pp. 781

Author(s):

Shoko Tokumoto ◽

Yugo Miyata ◽

Kengo Usui ◽

Ruslan Deviatiiarov ◽

Takahiro Ohkawa ◽

...

Keyword(s):

Cell Line ◽

Fluorescent Protein ◽

Expression System ◽

Constitutive Expression ◽

Reporter Genes ◽

Inducible Expression ◽

Minimal Promoter ◽

Luciferase Reporter ◽

Α Subunit ◽

Inducible Expression System

The Pv11 cell line established from an African chironomid, Polypedilum vanderplanki, is the only cell line tolerant to complete desiccation. In Pv11 cells, a constitutive expression system for Pv11 cells was previously exploited and several reporter genes were successfully expressed. Here we report the identification of an effective minimal promoter for Pv11 cells and its application to the Tet-On inducible expression system. First, using a luciferase reporter assay, we showed that a 202 bp deletion fragment derived from the constitutively active 121-promoter functions in Pv11 cells as an appropriate minimal promoter with the Tet-On inducible expression system. The AcGFP1 (Aequorea coerulescens green fluorescent protein) was also successfully expressed in Pv11 cells using the inducible system. In addition to these reporter genes, the avian myeloblastosis virus reverse transcriptase α subunit (AMV RTα), which is one of the most widely commercially available RNA-dependent DNA polymerases, was successfully expressed through the inducible expression system and its catalytic activity was verified. These results demonstrate the establishment of an inducible expression system in cells that can be preserved in the dry state and highlight a possible application to the production of large and complex proteins.

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Development and Application of an Arabinose-Inducible Expression System by Facilitating Inducer Uptake in Corynebacterium glutamicum

Applied and Environmental Microbiology ◽

10.1128/aem.01147-12 ◽

2012 ◽

Vol 78 (16) ◽

pp. 5831-5838 ◽

Cited By ~ 34

Author(s):

Yun Zhang ◽

Xiuling Shang ◽

Shujuan Lai ◽

Guoqiang Zhang ◽

Yong Liang ◽

...

Keyword(s):

Corynebacterium Glutamicum ◽

Fluorescent Protein ◽

Expression System ◽

Carbon Sources ◽

Inducible Expression ◽

Gene Encoding ◽

Content Type ◽

E Coli ◽

Inducible Expression System ◽

Green Fluorescent

ABSTRACTCorynebacterium glutamicumis currently used for the industrial production of a variety of biological materials. Many available inducible expression systems in this species uselac-derived promoters fromEscherichia colithat exhibit much lower levels of inducible expression and leaky basal expression. We developed an arabinose-inducible expression system that contains thel-arabinose regulator AraC, thePBADpromoter from thearaBADoperon, and thel-arabinose transporter AraE, all of which are derived fromE. coli. The level of induciblePBAD-based expression could be modulated over a wide concentration range from 0.001 to 0.4%l-arabinose. This system tightly controlled the expression of the uracil phosphoribosyltransferase without leaky expression. When the gene encoding green fluorescent protein (GFP) was under the control ofPBADpromoter, flow cytometry analysis showed that GFP was expressed in a highly homogeneous profile throughout the cell population. In contrast to the case inE. coli,PBADinduction was not significantly affected in the presence of different carbon sources inC. glutamicum, which makes it useful in fermentation applications. We used this system to regulate the expression of theodhIgene fromC. glutamicum, which encodes an inhibitor of α-oxoglutarate dehydrogenase, resulting in high levels of glutamate production (up to 13.7 mM) under biotin nonlimiting conditions. This system provides an efficient tool available for molecular biology and metabolic engineering ofC. glutamicum.

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An Inducible Expression System to Measure Rhodopsin Transport in Transgenic Xenopus Rod Outer Segments

PLoS ONE ◽

10.1371/journal.pone.0082629 ◽

2013 ◽

Vol 8 (12) ◽

pp. e82629 ◽

Cited By ~ 5

Author(s):

Xinming Zhuo ◽

Mohammad Haeri ◽

Eduardo Solessio ◽

Barry E. Knox

Keyword(s):

Expression System ◽

Inducible Expression ◽

Rod Outer Segments ◽

Inducible Expression System ◽

Outer Segments

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A new inducible expression system in a transformed green alga, Chlorella vulgaris

Genetics and Molecular Research ◽

10.4238/2011.october.21.1 ◽

2011 ◽

Vol 10 (4) ◽

pp. 3427-3434 ◽

Cited By ~ 40

Author(s):

Y.F. Niu ◽

M.H. Zhang ◽

W.H. Xie ◽

J.N. Li ◽

Y.F. Gao ◽

...

Keyword(s):

Green Alga ◽

Chlorella Vulgaris ◽

Expression System ◽

Inducible Expression ◽

Inducible Expression System ◽

Green Alga Chlorella

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Assembly of G Protein-Coupled Receptors onto Nanosized Bacterial Magnetic Particles Using Mms16 as an Anchor Molecule

Applied and Environmental Microbiology ◽

10.1128/aem.70.5.2880-2885.2004 ◽

2004 ◽

Vol 70 (5) ◽

pp. 2880-2885 ◽

Cited By ~ 49

Author(s):

Tomoko Yoshino ◽

Masayoshi Takahashi ◽

Haruko Takeyama ◽

Yoshiko Okamura ◽

Fukuichi Kato ◽

...

Keyword(s):

G Protein ◽

Magnetic Particles ◽

Lipid Membrane ◽

Specific Protein ◽

G Protein Coupled Receptors ◽

Native Conformation ◽

Wide Range ◽

Bacterial Magnetic Particles ◽

Magnetospirillum Magneticum ◽

G Protein Coupled

ABSTRACT G protein-coupled receptors (GPCRs) play a central role in a wide range of biological processes and are prime targets for drug discovery. GPCRs have large hydrophobic domains, and therefore purification of GPCRs from cells is frequently time-consuming and typically results in loss of native conformation. In this work, GPCRs have been successfully assembled into the lipid membrane of nanosized bacterial magnetic particles (BMPs) produced by the magnetic bacterium Magnetospirillum magneticum AMB-1. A BMP-specific protein, Mms16, was used as an anchor molecule, and localization of heterologous Mms16 on BMPs was confirmed by luciferase fusion studies. Stable luminescence was obtained from BMPs bearing Mms16 fused with luciferase at the C-terminal region. D1 dopamine receptor (D1R), a GPCR, was also efficiently assembled onto BMPs by using Mms16 as an anchor molecule. D1R-BMP complexes were simply extracted by magnetic separation from ruptured AMB-1 transformants. After washing, the complexes were ready to use for analysis. This system conveniently refines the native conformation of GPCRs without the need for detergent solubilization, purification, and reconstitution after cell disruption.

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