Expression of cloned genes and translation of messenger RNA in microinjected Xenopus oocytes

Heikkila, John J.

doi:10.1016/0020-711x(90)90302-j

“…To investigate chaperone activity in an in vivo homologous system, we developed a novel Xenopus oocyte assay system. Because this large cell (equivalent to 200 000 somatic cells) is amenable to microinjection, it has been used in a wide variety of assays, including transcription of DNA, translation of mRNA, analysis of trans-acting factors, and protein biochemistry (Heikkila 1990). Interestingly, LUC enzyme reactivation in the presence of 30C occurred more rapidly after microinjection into Xenopus oocytes than in the RRL system.…”

Section: Discussionmentioning

confidence: 99%

Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state

Abdulle

¹

,

Mohindra²,

Fernando³

et al. 2002

Self Cite

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In this study we characterized the chaperone functions of Xenopus recombinant Hsp30C and Hsp30D by using an in vitro rabbit reticulocyte lysate (RRL) refolding assay system as well as a novel in vivo Xenopus oocyte microinjection assay. Whereas heat- or chemically denaturated luciferase (LUC) did not regain significant enzyme activity when added to RRL or microinjected into Xenopus oocytes, compared with native LUC, denaturation of LUC in the presence of Hsp30C resulted in a reactivation of enzyme activity up to 80-100%. Recombinant Hsp30D, which differs from Hsp30C by 19 amino acids, was not as effective as its isoform in preventing LUC aggregation or maintaining it in a folding-competent state. Removal of the first 17 amino acids from the N-terminal region of Hsp30C had little effect on its ability to maintain LUC in a folding-competent state. However, deletion of the last 25 residues from the C-terminal end dramatically reduced Hsp30C chaperone activity. Coimmunoprecipitation and immunoblot analyses revealed that Hsp30C remained associated with heat-denatured LUC during incubation in reticulocyte lysate and that the C-terminal mutant exhibited reduced affinity for unfolded LUC. Finally, we found that Hsc70 present in RRL interacted only with heat-denatured LUC bound to Hsp30C. These findings demonstrate that Xenopus Hsp30 can maintain denatured target protein in a folding-competent state and that the C-terminal end is involved in this function.

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“…From a single-molecule perspective, subunit counting in oocytes has been the widely used approach, with many receptors being studied after controlled mRNA injection to limit receptor levels 18 . However, the concentration-dependence of oligomerization may be at odds with the sub-physiological expression levels employed in this technique and cell type specific post-translational modifications occurring in the Golgi and ER required for native oligomer formation may be lacking 19 . We show that cell fusion combined with single molecule imaging lifts this restriction and allows single molecule imaging after physiological assembly of receptor complexes in a cell type required by any specific biological constraints.…”

Section: Dilution Of Labelled Cytosolic Proteins By Cell Fusion Impromentioning

confidence: 99%

Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging

Singh

¹

,

Slyke

²

,

Sirenko

³

et al. 2020

Sci Rep

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The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.

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“…From a single-molecule perspective, subunit counting in oocytes has been the widely used approach, with many receptors being studied after controlled mRNA injection to limit receptor levels (16). However, the concentration-dependence of oligomerization may be at odds with the sub-physiological expression levels employed in this technique and cell type specific post-translational modifications occurring in the Golgi and ER required for native oligomer formation may be lacking (17). We show that cell fusion combined with single molecule imaging lifts this restriction and allows single molecule imaging after physiological assembly of receptor complexes in a cell type required by any specific biological constraints.…”

Section: Single Molecule Imaging Of Membrane Protein Complexesmentioning

confidence: 99%

Stoichiometric Analysis of Protein Complexes by Cell Fusion and Single Molecule Imaging

Singh

¹

,

Slyke

²

,

Sirenko

³

et al. 2020

Preprint

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The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry. SIGNIFICANCE STATEMENTQuantifying the composition and stoichiometry of protein complexes in live cells is critical to understanding mechanisms involved in their function. Here we detail a method in which protein complexes are assembled intracellularly at physiological concentrations, but then diluted to levels suitable for single-molecule fluorescence observations while still within a cellular environment. The technique permits the use of common single molecule analysis techniques such as stepwise photobleaching quantification and fluorescence correlation spectroscopy to determine stoichiometry and functional interactions while avoiding artifacts that may occur from the use of detergent isolation methods or from the artificially low expression levels sometimes used to attain single molecule observation levels.

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Expression of cloned genes and translation of messenger RNA in microinjected Xenopus oocytes

Cited by 11 publications

References 57 publications

Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state

Xenopus small heat shock proteins, Hsp30C and Hsp30D, maintain heat- and chemically denatured luciferase in a folding-competent state

Stoichiometric analysis of protein complexes by cell fusion and single molecule imaging

Stoichiometric Analysis of Protein Complexes by Cell Fusion and Single Molecule Imaging

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