Incorporation of Selenomethionine into Induced Intracytoplasmic Membrane Proteins of Rhodobacter species

Laible, Philip D.; Hata, Aaron N.; Crawford, Adam E; Hanson, Deborah K.

doi:10.1007/s10969-005-1936-3

Cited by 8 publications

(3 citation statements)

References 32 publications

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“…PUC705-BA bearing the empty pRKPLHT7 plasmid was used as a negative control in expression screening with western-blot. Unless otherwise noted, R. sphaeroides strains were cultivated in minimal (MR26) [30] or complex medium (YCC) [31] under semi-aerobic or photoheterotrophic conditions with 1 µg/mL tetracycline as required for selection [17]. Variations in antibiotic use are described in a subsequent section.…”

Section: Methodsmentioning

confidence: 99%

Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides

et al. 2014

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Aquaporins are highly selective water channel proteins integrated into plasma membranes of single cell organisms; plant roots and stromae; eye lenses, renal and red blood cells in vertebrates. To date, only a few microbial aquaporins have been characterized and their physiological importance is not well understood. Here we report on the cloning, expression and characterization of a novel aquaporin, RsAqpZ, from a purple photosynthetic bacterium, Rhodobacter sphaeroides ATCC 17023. The protein was expressed homologously at a high yield (∼20 mg/L culture) under anaerobic photoheterotrophic growth conditions. Stopped-flow light scattering experiments demonstrated its high water permeability (0.17±0.05 cm/s) and low energy of activation for water transport (2.93±0.60 kcal/mol) in reconstituted proteoliposomes at a protein to lipid ratio (w/w) of 0.04. We developed a fluorescence correlation spectroscopy based technique and utilized a fluorescent protein fusion of RsAqpZ, to estimate the single channel water permeability of RsAqpZ as 1.24 (±0.41) x 10−12 cm3/s or 4.17 (±1.38)×1010 H2O molecules/s, which is among the highest single channel permeability reported for aquaporins. Towards application to water purification technologies, we also demonstrated functional incorporation of RsAqpZ in amphiphilic block copolymer membranes.

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Section: Methodsmentioning

confidence: 99%

Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides

et al. 2014

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“…Among prokaryotic hosts, the photosynthetic bacteria Rhodobacter sphaeroides and Rhodobacter capsulatus provide plenty of membrane space while concomitantly offering elaborate and highly efficient insertion machineries. During prototrophic growth, they use light for energy production and growth, and thereby enrich intracytoplasmic membranes [63-65]. However future experiments will have to demonstrate whether this is sufficient to obtain high yields of membrane proteins.…”

Section: Reviewmentioning

confidence: 99%

Tuning microbial hosts for membrane protein production

2009

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The last four years have brought exciting progress in membrane protein research. Finally those many efforts that have been put into expression of eukaryotic membrane proteins are coming to fruition and enable to solve an ever-growing number of high resolution structures. In the past, many skilful optimization steps were required to achieve sufficient expression of functional membrane proteins. Optimization was performed individually for every membrane protein, but provided insight about commonly encountered bottlenecks and, more importantly, general guidelines how to alleviate cellular limitations during microbial membrane protein expression. Lately, system-wide analyses are emerging as powerful means to decipher cellular bottlenecks during heterologous protein production and their use in microbial membrane protein expression has grown in popularity during the past months.This review covers the most prominent solutions and pitfalls in expression of eukaryotic membrane proteins using microbial hosts (prokaryotes, yeasts), highlights skilful applications of our basic understanding to improve membrane protein production. Omics technologies provide new concepts to engineer microbial hosts for membrane protein production.

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“…MSCG is in the process of expanding the approach to well-expressed but insoluble proteins [8] and membrane proteins [9].…”

Section: Introductionmentioning

confidence: 99%

Automation of protein purification for structural genomics

Kim

Dementieva

Zhou

et al. 2004

J Struct Func Genom

103

108

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A critical issue in structural genomics, and in structural biology in general, is the availability of highquality samples. The additional challenge in structural genomics is the need to produce high numbers of proteins with low sequence similarities and poorly characterized or unknown properties. 'Structural-biology-grade' proteins must be generated in a quantity and quality suitable for structure determination experiments using X-ray crystallography or nuclear magnetic resonance (NMR). The choice of protein purification and handling procedures plays a critical role in obtaining high-quality protein samples. The purification procedure must yield a homogeneous protein and must be highly reproducible in order to supply milligram quantities of protein and/or its derivative containing marker atom(s). At the Midwest Center for Structural Genomics we have developed protocols for highthroughput protein purification. These protocols have been implemented on AKTA EXPLORER 3D and AKTA FPLC 3D workstations capable of performing multidimensional chromatography. The automated chromatography has been successfully applied to many soluble proteins of microbial origin. Various MCSG purification strategies, their implementation, and their success rates are discussed in this paper.

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Incorporation of Selenomethionine into Induced Intracytoplasmic Membrane Proteins of Rhodobacter species

Cited by 8 publications

References 32 publications

Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides

Molecular Cloning, Overexpression and Characterization of a Novel Water Channel Protein from Rhodobacter sphaeroides

Tuning microbial hosts for membrane protein production

Automation of protein purification for structural genomics

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