Expression, purification and properties of multidrug efflux proteins

2000 ◽  
Vol 28 (4) ◽  
pp. 513-517 ◽  
Author(s):  
P.J. F. Henderson ◽  
C. K. Hoyle ◽  
A. Ward
Keyword(s):  
Fluorescent Protein ◽  
Affinity Column ◽  
General Strategy ◽  
X Rays ◽  
Multidrug Efflux ◽  
Nmr Studies ◽  
E Coli ◽  
Efflux Proteins

A general strategy is described for the amplified expression, purification and characterization in Escherichia coli of multidrug efflux proteins from Staphylococcus aureus, Bacillus subtilis, Methanococcus janaschii and E. coli. They all catalyse drug/H+ antiport of substrates such as quinolones and ethidium and exemplify a family of putatively 12-helix membrane proteins. The gene for each protein was cloned downstream of the tac promoter in plasmid pTTQ18; an oligonucleotide encoding six histidine residues was added, in frame, to the C-terminus to facilitate purification. Growth conditions were optimized in 1–25-litre cultures of E. coli host strains to amplify the expression of each protein; the retention of activity was confirmed by assays of antibiotic resistance in vivo and/or assays of energized transport activity in vitro with synthetic substrates. Proteins were solubilized in dodecylmaltoside and purified to more than 90% homogeneity with Ni2+-nitrilo-triacetate-affinity column chromography, yielding 5–25 mg per 25 litres of original culture. All the transport proteins migrated anomalously in SDS/PAGE at apparent molecular masses below those predicted from the gene sequence; identity and integrity were therefore confirmed by N-terminal amino acid sequencing and Western blotting for the C-terminal hexahistidine tag. Examination of the secondary structure of detergent-solubilized proteins by CD or Fourier-transform infrared spectroscopy following purification indicated a high content of α-helix (more than 75%). Matrix-assisted laser desorption ionization MS confirmed the high degree of purity and the true molecular mass. The formation of three-dimensional crystals is being attempted but crystals have yet to be grown that diffract X-rays. The growth of two-dimensional protein arrays has been more successful, with diffraction of electrons at low resolution. Proteins have been fused to green fluorescent protein or maltose-binding protein to facilitate these structural analyses. In addition, ligands for efflux proteins labelled with 13C or 15N have been synthesized to implement solid-state NMR studies of the ligand-binding site.

scholarly journals Direct preparation of Cas9 ribonucleoprotein from E. coli for PCR-free seamless DNA assembly

2018 ◽  
Author(s):  
Wenqiang Li ◽  
Shuntang Li ◽  
Jie Qiao ◽  
Fei Wang ◽  
Yang Liu ◽  
...  
Keyword(s):  
Large Scale ◽  
Genome Engineering ◽  
Restriction Enzymes ◽  
General Strategy ◽  
Dna Assembly ◽  
E Coli ◽  
Assembly Method ◽  
Direct Preparation

AbstractCRISPR-Cas9 is a versatile and powerful genome engineering tool. Recently, Cas9 ribonucleoprotein (RNP) complexes have been used as promising biological tools with plenty of in vivo and in vitro applications, but there are by far no efficient methods to produce Cas9 RNP at large scale and low cost. Here, we describe a simple and effective approach for direct preparation of Cas9 RNP from E. coli by co-expressing Cas9 and target specific single guided RNAs. The purified RNP showed in vivo genome editing ability, as well as in vitro endonuclease activity that combines with an unexpected superior stability to enable routine uses in molecular cloning instead of restriction enzymes. We further develop a RNP-based PCR-free method termed Cas-Brick in a one-step or cyclic way for seamless assembly of multiple DNA fragments with high fidelity up to 99%. Altogether, our findings provide a general strategy to prepare Cas9 RNP and supply a convenient and cost-effective DNA assembly method as an invaluable addition to synthetic biological toolboxes.

Survival Analysis of Escherichia coli Encapsulated in a Hollow Fiber Membrane In Vitro and In Vivo: Preliminary Report

2005 ◽  
Vol 14 (5) ◽  
pp. 323-330 ◽  
Author(s):  
L. H. Granicka ◽  
M. Wdowiak ◽  
A. Kosek ◽  
S. Świezewski ◽  
D. Wasilewska ◽  
...  
Keyword(s):  
Hollow Fiber ◽  
Fluorescent Protein ◽  
Culture Media ◽  
Hollow Fiber Membrane ◽  
In Vitro Experiments ◽  
Bacteria Growth ◽  
E Coli ◽  
Green Fluorescent

The purpose of the observations was the viability and quality evaluation of E. coli bacteria encapsulated in hollow fiber membranes (HF) in short in vivo and in vitro experiments. A polypropylene, surface-modified hollow fiber was applied for immunoisolation of E. coli bacteria transfected with a green fluorescent protein (E. coli GFPI). The presence of GFP fluorescence of organisms was assessed with the use of flow cytometry. The E. coli GFPIs were then observed for the period of 5 days in in vitro experiments in the culture medium. A single IPTG (isopropyl β-D-1-thiogalactopyranoside) induction of GFP gene appeared to be adequate for an expression of GFP protein for 5 days. The GFP expression values observed for E. coli GFPs encapsulated in HF during culture in different culture media were comparable. The survival of E. coli GFPIs encapsulated in HF after 1, 2, 4, or 5 days of subcutaneous implantation into mice was evaluated. The explanted E. coli GFPIs exhibited mean expression 603 ± 17 (n = 32) units of fluorescence during the implantation period. The values obtained were comparable for selected days of observation. It was observed that the membranes applied ensured the bacteria growth within the HF's space only.

scholarly journals The Tubulin-Like RepX Protein Encoded by the pXO1 Plasmid Forms Polymers In Vivo in Bacillus anthracis

2009 ◽  
Vol 191 (8) ◽  
pp. 2493-2500 ◽  
Author(s):  
Parvez Akhtar ◽  
Syam P. Anand ◽  
Simon C. Watkins ◽  
Saleem A. Khan
Keyword(s):  
Bacillus Anthracis ◽  
Cell Morphology ◽  
Fluorescent Protein ◽  
Host Cells ◽  
E Coli ◽  
Green Fluorescent ◽  
Related Proteins ◽  
Polymeric Structures

ABSTRACT Bacillus anthracis contains two megaplasmids, pXO1 and pXO2, that are critical for its pathogenesis. Stable inheritance of pXO1 in B. anthracis is dependent upon the tubulin/FtsZ-like RepX protein encoded by this plasmid. Previously, we have shown that RepX undergoes GTP-dependent polymerization in vitro. However, the polymerization properties and localization pattern of RepX in vivo are not known. Here, we utilize a RepX-green fluorescent protein (GFP) fusion to show that RepX forms foci and three distinct forms of polymeric structures in B. anthracis in vivo, namely straight, curved, and helical filaments. Polymerization of RepX-GFP as well as the nature of polymers formed were dependent upon concentration of the protein inside the B. anthracis cells. RepX predominantly localized as polymers that were parallel to the length of the cell. RepX also formed polymers in Escherichia coli in the absence of other pXO1-encoded products, showing that in vivo polymerization is an inherent property of the protein and does not require either the pXO1 plasmid or proteins unique to B. anthracis. Overexpression of RepX did not affect the cell morphology of B. anthracis cells, whereas it drastically distorted the cell morphology of E. coli host cells. We discuss the significance of our observations in view of the plasmid-specific functions that have been proposed for RepX and related proteins encoded by several megaplasmids found in members of the Bacillus cereus group of bacteria.

scholarly journals Role of bacteriophages in STEC infections: new implications for the design of prophylactic and treatment approaches

F1000Research ◽  
2014 ◽  
Vol 3 ◽  
pp. 74 ◽  
Author(s):  
Jaime H. Amorim ◽  
Monica R. Jesus ◽  
Wilson B. Luiz ◽  
Bruna F.M.M. Porchia ◽  
Rita C.C. Ferreira ◽  
...  
Keyword(s):  
Shiga Toxin ◽  
Fluorescent Protein ◽  
Imaging System ◽  
Lytic Replication ◽  
Biologically Active ◽  
Gene Encoding ◽  
E Coli ◽  
Green Fluorescent

Shiga toxin (Stx) is considered the main virulence factor in Shiga toxin-producing Escherichia coli (STEC) infections. Previously we reported the expression of biologically active Stx by eukaryotic cells in vitro and in vivo following transfection with plasmids encoding Stx under control of the native bacterial promoter. Since stx genes are present in the genome of lysogenic bacteriophages, here we evaluated the relevance of bacteriophages during STEC infection. We used the non-pathogenic E. coli K12 strain carrying a lysogenic 933W mutant bacteriophage in which the stx operon was replaced by a gene encoding the green fluorescent protein (GFP). Tracking GFP expression using an In Vivo Imaging System (IVIS), we detected fluorescence in liver, kidney, and intestine of mice infected with the recombinant E. coli strain after treatment with ciprofloxacin, which induces the lytic replication and release of bacteriophages. In addition, we showed that chitosan, a linear polysaccharide composed of D-glucosamine residues and with a number of commercial and biomedical uses, had strong anti-bacteriophage effects, as demonstrated in vitro and in vivo. These findings bring promising perspectives for the prevention and treatment of hemolytic uremic syndrome (HUS) cases.

Rapid, Refined, and Robust Method for Expression, Purification, and Characterization of Recombinant Human Amyloid-beta M1-42

2019 ◽  
Author(s):  
Priya Prakash ◽  
Travis Lantz ◽  
Krupal P. Jethava ◽  
Gaurav Chopra
Keyword(s):  
Amyloid Beta ◽  
Western Blots ◽  
Force Microscopy ◽  
E Coli ◽  
Atomic Force ◽  
Set Up ◽  
Short Time

Amyloid plaques found in the brains of Alzheimer’s disease (AD) patients primarily consists of amyloid beta 1-42 (Ab42). Commercially, Ab42 is synthetized using peptide synthesizers. We describe a robust methodology for expression of recombinant human Ab(M1-42) in Rosetta(DE3)pLysS and BL21(DE3)pLysS competent E. coli with refined and rapid analytical purification techniques. The peptide is isolated and purified from the transformed cells using an optimized set-up for reverse-phase HPLC protocol, using commonly available C18 columns, yielding high amounts of peptide (~15-20 mg per 1 L culture) in a short time. The recombinant Ab(M1-42) forms characteristic aggregates similar to synthetic Ab42 aggregates as verified by western blots and atomic force microscopy to warrant future biological use. Our rapid, refined, and robust technique to purify human Ab(M1-42) can be used to synthesize chemical probes for several downstream in vitro and in vivo assays to facilitate AD research.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Radiosensitization of Prostate Cancers In Vitro and In Vivo to Erbium-filtered Orthovoltage X-rays Using Actively Targeted Gold Nanoparticles

2017 ◽  
Vol 7 (1) ◽  
Author(s):  
Allison M. Khoo ◽  
Sang Hyun Cho ◽  
Francisco J. Reynoso ◽  
Maureen Aliru ◽  
Kathryn Aziz ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
X Rays ◽  
Prostate Cancers

scholarly journals Optimization of X-ray Investigations in Dentistry Using Optical Coherence Tomography

Sensors ◽  
2021 ◽  
Vol 21 (13) ◽  
pp. 4554
Author(s):  
Ralph-Alexandru Erdelyi ◽  
Virgil-Florin Duma ◽  
Cosmin Sinescu ◽  
George Mihai Dobre ◽  
Adrian Bradu ◽  
...  
Keyword(s):  
Optical Coherence Tomography ◽  
Radiation Dose ◽  
Imaging Techniques ◽  
Image Resolution ◽  
Optical Coherence ◽  
X Rays ◽  
High Quality ◽  
X Ray

The most common imaging technique for dental diagnoses and treatment monitoring is X-ray imaging, which evolved from the first intraoral radiographs to high-quality three-dimensional (3D) Cone Beam Computed Tomography (CBCT). Other imaging techniques have shown potential, such as Optical Coherence Tomography (OCT). We have recently reported on the boundaries of these two types of techniques, regarding. the dental fields where each one is more appropriate or where they should be both used. The aim of the present study is to explore the unique capabilities of the OCT technique to optimize X-ray units imaging (i.e., in terms of image resolution, radiation dose, or contrast). Two types of commercially available and widely used X-ray units are considered. To adjust their parameters, a protocol is developed to employ OCT images of dental conditions that are documented on high (i.e., less than 10 μm) resolution OCT images (both B-scans/cross sections and 3D reconstructions) but are hardly identified on the 200 to 75 μm resolution panoramic or CBCT radiographs. The optimized calibration of the X-ray unit includes choosing appropriate values for the anode voltage and current intensity of the X-ray tube, as well as the patient’s positioning, in order to reach the highest possible X-rays resolution at a radiation dose that is safe for the patient. The optimization protocol is developed in vitro on OCT images of extracted teeth and is further applied in vivo for each type of dental investigation. Optimized radiographic results are compared with un-optimized previously performed radiographs. Also, we show that OCT can permit a rigorous comparison between two (types of) X-ray units. In conclusion, high-quality dental images are possible using low radiation doses if an optimized protocol, developed using OCT, is applied for each type of dental investigation. Also, there are situations when the X-ray technology has drawbacks for dental diagnosis or treatment assessment. In such situations, OCT proves capable to provide qualitative images.

scholarly journals Arabidopsis Disulfide Reductase, Trx-h2, Functions as an RNA Chaperone under Cold Stress

2021 ◽  
Vol 11 (15) ◽  
pp. 6865
Author(s):  
Eun Seon Lee ◽  
Joung Hun Park ◽  
Seong Dong Wi ◽  
Ho Byoung Chae ◽  
Seol Ki Paeng ◽  
...  
Keyword(s):  
Cold Stress ◽  
Wild Type ◽  
Rna Chaperone ◽  
E Coli ◽  
Cold Sensitive ◽  
Thioredoxin H ◽  
Functional Rnas

The thioredoxin-h (Trx-h) family of Arabidopsis thaliana comprises cytosolic disulfide reductases. However, the physiological function of Trx-h2, which contains an additional 19 amino acids at its N-terminus, remains unclear. In this study, we investigated the molecular function of Trx-h2 both in vitro and in vivo and found that Arabidopsis Trx-h2 overexpression (Trx-h2OE) lines showed significantly longer roots than wild-type plants under cold stress. Therefore, we further investigated the role of Trx-h2 under cold stress. Our results revealed that Trx-h2 functions as an RNA chaperone by melting misfolded and non-functional RNAs, and by facilitating their correct folding into active forms with native conformation. We showed that Trx-h2 binds to and efficiently melts nucleic acids (ssDNA, dsDNA, and RNA), and facilitates the export of mRNAs from the nucleus to the cytoplasm under cold stress. Moreover, overexpression of Trx-h2 increased the survival rate of the cold-sensitive E. coli BX04 cells under low temperature. Thus, our data show that Trx-h2 performs function as an RNA chaperone under cold stress, thus increasing plant cold tolerance.

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