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Related Concept Videos

Conservation of Protein Domains Over Different Proteins02:26

Conservation of Protein Domains Over Different Proteins

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Protein domains are small structurally independent units that are part of a single amino acid chain.  Although these domains are often structurally independent, they may rely on synergistic effects to perform their functions as part of a larger protein. Protein domains may be conserved within the same organism, as well as across different organisms.
A limited set of protein domains often duplicate and recombine during evolution. These domains can be organized in different combinations to...
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Conservation of Protein Domains02:26

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Solubility is the measure of the maximum amount of solute that can be dissolved in a given quantity of solvent at a given temperature and pressure. Solubility is usually measured in molarity (M) or moles per liter (mol/L). A compound is termed soluble if it dissolves in water.
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Compared with pure water, the solubility of an ionic compound is less in aqueous solutions containing a common ion (one also produced by dissolution of the ionic compound). This is an example of a phenomenon known as the common ion effect, which is a consequence of the law of mass action that may be explained using Le Chȃtelier’s principle. Consider the dissolution of silver iodide:
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Solubility Equilibria03:07

Solubility Equilibria

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Solubility equilibria are established when the dissolution and precipitation of a solute species occur at equal rates. These equilibria underlie many natural and technological processes, ranging from tooth decay to water purification. An understanding of the factors affecting compound solubility is, therefore, essential to the effective management of these processes. This section applies previously introduced equilibrium concepts and tools to systems involving dissolution and precipitation.
The...
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Physical Properties Affecting Solubility02:19

Physical Properties Affecting Solubility

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Solutions of Gases in Liquids
As for any solution, the solubility of a gas in a liquid is affected by the attractive intermolecular forces between solute and solvent species. Unlike solid and liquid solutes, however, there is no solute-solute intermolecular attraction to overcome when a gaseous solute dissolves in a liquid solvent since the atoms or molecules comprising a gas are far separated and experience negligible interactions. Consequently, solute-solvent interactions are the sole...
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Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration
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High-Throughput Isolation of Soluble Protein Domains Using a Bipartite Split-GFP Complementation System.

Amélie Massemin1, Stéphanie Cabantous2, Geoffrey S Waldo3

  • 1Institut de Pharmacologie et de Biologie Structurale, IPBS, Université de Toulouse, CNRS, UPS, Toulouse, France.

Methods in Molecular Biology (Clifton, N.J.)
|July 4, 2019
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Summary

We developed a split-green fluorescent protein (split-GFP) assay to identify soluble, folded protein domains. This method uses self-assembling GFP fragments for efficient protein domain identification in E. coli.

Keywords:
Domain trappingProtein fragment complementationProtein solubilityProtein taggingSplit-GFP

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Protein Engineering

Background:

  • Identifying soluble, folded protein domains is crucial in molecular biology.
  • Current methods may lack efficiency or require specific conditions.

Purpose of the Study:

  • To present a protocol for identifying compact, soluble protein domains.
  • To utilize a novel self-assembling split-green fluorescent protein (split-GFP) system.

Main Methods:

  • A two-part split-GFP system was employed, consisting of GFP1-10 (detector fragment) and GFP11 (tagging fragment).
  • The assay was performed in E. coli cells and cell extracts.
  • Selection steps ensured correct protein fragment assembly (in-frame, no stop codons).
  • Inverse PCR was used to enrich libraries with specific target sequences.

Main Results:

  • The developed protocol effectively identifies soluble, folded protein domains.
  • The split-GFP system demonstrated successful self-assembly and detection.
  • The selection and enrichment steps improved the efficiency of identifying target protein fragments.

Conclusions:

  • This split-GFP based protocol offers a robust method for identifying soluble protein domains.
  • The assay is adaptable for use in various E. coli-based expression systems.
  • This technique facilitates protein engineering and structural biology studies.