A green fluorescent protein screen for identification of well‐expressed membrane proteins from a cohort of extremophilic organisms

Hammon, Justus; Palanivelu, Dinesh V.; Chen, Joy; Patel, Chintan K.; Minor, Daniel L.

doi:10.1002/pro.18

Cited by 53 publications

(45 citation statements)

References 53 publications

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“…Building upon previously described reports using GFP as a reporter for monitoring expression and purification, [3][4][5][6][7][8][9][10][14][15][16] we established a comprehensive, streamlined approach for confirming topology, optimizing expression, and screening stable protein-detergent complexes for both C in and C out membrane proteins. The pWarf vector system consists of two vectors, pWarf(À) and pWarf(þ), that were constructed by modifying the pET28 vector described in Drew et al 9 (Supporting Information Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Several studies have employed GFP as a reporter for membrane protein expression and detergent selection, [3][4][5][6]9,10,14,15 but the technology has largely been restricted to proteins with intracellular C-termini (C in ) due to the GFP's inability to mature when localized to the E. coli periplasm. 11 We have demonstrated that Glycophorin A (GpA)-a single spanning transmembrane helix-effectively converts C out membrane proteins to become C in proteins where the GFP can properly fold and fluoresce.…”

Section: Discussionmentioning

confidence: 99%

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Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

et al. 2010

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Low expression and instability during isolation are major obstacles preventing adequate structure-function characterization of membrane proteins (MPs). To increase the likelihood of generating large quantities of protein, C-terminally fused green fluorescent protein (GFP) is commonly used as a reporter for monitoring expression and evaluating purification. This technique has mainly been restricted to MPs with intracellular C-termini (C in ) due to GFP's inability to fluoresce in the Escherichia coli periplasm. With the aid of Glycophorin A, a single transmembrane spanning protein, we developed a method to convert MPs with extracellular C-termini (C out ) to C in ones providing a conduit for implementing GFP reporting. We tested this method on eleven MPs with predicted C out topology resulting in high level expression. For nine of the eleven MPs, a stable, monodisperse protein-detergent complex was identified using an extended fluorescencedetection size exclusion chromatography procedure that monitors protein stability over time, a critical parameter affecting the success of structure-function studies. Five MPs were successfully cleaved from the GFP tag by site-specific proteolysis and purified to homogeneity. To address the challenge of inefficient proteolysis, we explored expression and purification conditions in the absence of the fusion tag. Contrary to previous studies, optimal expression conditions established with the fusion were not directly transferable for overexpression in the absence of the GFP tag. These studies establish a broadly applicable method for GFP screening of MPs with C out topology, yielding sufficient protein suitable for structure-function studies and are superior to expression and purification in the absence GFP fusion tagging.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

et al. 2010

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“…To that end, this protocol can further be adapted for purification of recombinant GFP-fusion proteins, in which an unrelated target protein is "tagged" with GFP. GFP-tagged target proteins can be detected with UV light 11 and purified using various chromatographic approaches, including the HIC purification strategy described above. GFP purification has also become a pedagogical staple in biochemistry labs for the teaching of modern protein science techniques 12 .…”

Section: Discussionmentioning

confidence: 99%

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Murphy

Stone

Anderson

2011

JoVE

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In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1 . The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH 4 ) 2 SO 4 ). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2 . As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in prepacked columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3 . Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4 .The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5 . Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

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“…Measurement of GFP Relative Fluorescence Intensity in E. coli GFP-fusion fluorescence intensity was an excellent indicator of over-expression potential [30]. Because fluorescence was one of the most convenient ways to follow a protein expression and purification procedure [31], so the fluorescence intensity was used to analyze the expression efficiency of proteins.…”

Section: A Constructions Of Plasmid and Cloning Vectorsmentioning

confidence: 99%

Influence of Some Point Mutations of Green Fluorescent Protein Gene on Expression Efficiency in Escherichia coli

Hao¹

2016

IJCEA

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Abstract-In this paper, I studied on the influence of some point mutations of green fluorescent protein (GFP) gene on expression efficiency in Escherichia coli (E. coli). It was found that some mutant genes of GFP had influenced on the expression efficiency of GFP gene. In this paper, I acquired six GFP mutants (A402G, A675C, A462G, A88G, T357C, A443G), compared the differences among the six mutant sites, and discussed the relationship between the mutant gene and protein expression efficiency. It was found that A443G had the lowest relative fluorescence intensity (0.04 fold), and A88G had the lower relative fluorescence intensity (0.16 fold), A402G had the higher relative fluorescence intensity (1.2 fold), against wildtype of GFP. I hope that the findings, which may be applicable to genetic engineering, will be helpful for further studies of protein expression.

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A green fluorescent protein screen for identification of well‐expressed membrane proteins from a cohort of extremophilic organisms

Cited by 53 publications

References 53 publications

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

Bridging the gap: A GFP‐based strategy for overexpression and purification of membrane proteins with intra and extracellular C‐termini

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

Influence of Some Point Mutations of Green Fluorescent Protein Gene on Expression Efficiency in Escherichia coli

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