A novel chromatography system to isolate active ribosomes from pathogenic bacteria

Maguire, Bruce A.; Wondrack, L; Contillo, Leonard G.; Xu, Zuoyu

doi:10.1261/rna.692408

Cited by 29 publications

(34 citation statements)

References 17 publications

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“…These concerns led us to investigate a chromatographic protocol employing a cysteine charged sulfolink resin previously used for purifying bacterial ribosomes from clinical isolates. 11 A flowchart of the traditional and chromatographic methods is shown in Figure 1.…”

Section: Resultsmentioning

confidence: 99%

“…10 The high levels of proteases and nucleases present in clinical isolates of pathogenic bacteria led Maguire and co-workers to devise a chromatographic method for ribosome purification using a cysteine charged Sulfolink resin. 11 The rRNAs and proteins derived from bacterial ribosomes isolated using this method showed much lower levels of degradation, and the ribosomes so purified were significantly more able to bind erythromycin and to synthesize proteins. These observations suggested that the cysteine charged sulfolink resin chromatography method may also be applicable to yeast ribosomes, and if so, that it could solve many of the problems described above.…”

Section: Introductionmentioning

confidence: 92%

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Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method

Leshin

Rakauskaitė²,

Dinman³

et al. 2010

RNA Biology

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One of the major challenges facing researchers working with eukaryotic ribosomes lies in their lability relative to their eubacterial and archael counterparts. In particular, lysis of cells and purification of eukaryotic ribosomes by conventional differential ultracentrifugation methods exposes them for long periods of time to a wide range of co-purifying proteases and nucleases, negatively impacting their structural integrity and functionality. A chromatographic method using a cysteine charged Sulfolink resin was adapted to address these problems. This fast and simple method significantly reduces co-purifying proteolytic and nucleolytic activities, producing good yields of highly biochemically active yeast ribosomes with fewer nicks in their rRNAs. In particular, the chromatographic purification protocol significantly improved the quality of ribosomes isolated from mutant cells. This method is likely applicable to mammalian ribosomes as well. The simplicity of the method, and the enhanced purity and activity of chromatographically purified ribosome represents a significant technical advancement for the study of eukaryotic ribosomes.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 92%

Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method

Leshin

Rakauskaitė²,

Dinman³

et al. 2010

RNA Biology

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show abstract

“…This protocol is based on Maguire et al (2008). A detailed chromatographic protocol for purifying ribosomes from S. cerevisiae has also been published (Leshin et al 2010), and ribosome-isolation protocols for higher organisms using similar methods are likely to follow.…”

Section: Related Informationmentioning

confidence: 99%

Isolation of Ribosomes by Chromatography

Maguire

2015

Cold Spring Harb Protoc

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Mixed-mode chromatography on cysteine-SulfoLink resin efficiently separates ribosomes from cell lysates and is particularly effective at rapidly removing endogenous proteases and nucleases, resulting in ribosomes of improved purity, integrity, and activity. Binding occurs partly by anion exchange of the RNA of the ribosomes, so that cells must be lysed in a buffer of moderate ionic strength (conductivity no more than 20 mS for chromatography of bacterial ribosomes) without any highly charged additives (e.g., heparin, which is used to inhibit RNases in yeast). A robust protocol for Escherichia coli is given here as an example.

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“…There is need of a method that ensures rapid isolation of active ribosomes from bacteria without the use of harsh conditions or lengthy procedures that damage ribosomes. Maguire et al (2008) developed a novel chromatography method that ensures that ribosomes obtained are intact and active when extracted from Escherichia coli, Deinococcus radiodurans, and some clinical isolates of Streptococcus pneumoniae and methicillin-resistant Staphylococcus aureus (MRSA). The system employs cysteine Sulfolink resin and shows unique features compared to other chromatography methods used for ribosomes or RNA.…”

Section: Ribosomesmentioning

confidence: 99%

“…Organic solvent-phenol-chloroform extraction is an example which is widely used in isolating nucleic acid from samples before electrophoresis on a Novex 6% acrylamide TBE/urea gel e.g. Invitrogen, Inc. for 100 min at 180V (Maguire et al, 2008). The electrophoresed gels are then stained with ethidium bromide for detection of RNA by fluorescence.…”

Section: Nucleic Acids (Dna and Rna)mentioning

confidence: 99%

How to obtain the organelles of prokaryotic and microbial eukaryotic cells

Aderiye¹,

Oluwole²

2014

J. Cell Anim. Biol.

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An organelle is a specialized functional subunit within cells carrying out specific functions. These compartments which may or may not be enclosed in a lipid bilayer are found in microorganisms. While those found in eukaryotic cells are usually enclosed in lipid bilayer, those in prokaryotes don't. All microbes have compartments common to them like the nucleic acids, protein, ribosomes as well as unique intracellular structures found only in microbial subgroups. Such compartments include the mitochondria, endoplasmic reticulum, golgi apparatus amongst others unique to all eukaryotic cells only. Prokaryotes contain some micro-compartments unique to them including the carboxysomes, lipid bodies, polyhydroxybutyrate granules. The right choice of cell disruption methods that limit damage to the compartments is important in achieving successful compartment isolation and purification. Commonly applied methods include sonication, enzymatic lysis, detergent lysis, cavitation amongst others depending on the type of cells involved. Fractionation is the commonly utilized method for isolation and purification of organelles, utilizing ultracentrifugation and techniques that exploits size, density and surface charge variations of protoplasmic content. Such techniques include gradient centrifugation methods, use of beads, affinity purification chromatography methods and electrophoresis. Here, we review the compartments in microbial cells and the techniques employed to isolate and purify these intracellular components.

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A novel chromatography system to isolate active ribosomes from pathogenic bacteria

Cited by 29 publications

References 17 publications

Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method

Enhanced purity, activity and structural integrity of yeast ribosomes purified using a general chromatographic method

Isolation of Ribosomes by Chromatography

How to obtain the organelles of prokaryotic and microbial eukaryotic cells

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