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Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli
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Normalized Screening of Protein Engineering Libraries by Split-GFP Crude Cell Extract Quantification.

Javier Santos-Aberturas1, Mark Dörr2, Uwe T Bornscheuer3

  • 1Department of Molecular Microbiology, John Innes Centre, Colney Lane, Norwich, UK.

Methods in Molecular Biology (Clifton, N.J.)
|November 1, 2017
PubMed
Summary
This summary is machine-generated.

Screening protein variants is challenging due to varying expression and solubility. Split-green fluorescent protein (GFP) technology normalizes activity by protein content, improving mutant identification for better protein engineering.

Keywords:
Data normalizationDirected evolutionHigh-throughput screeningMutant libraryProtein engineeringProtein solubilitySplit-GFP

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Area of Science:

  • Biotechnology
  • Protein Engineering
  • Molecular Biology

Background:

  • Protein variant screening is often limited by variations in expression levels and solubility.
  • Relying solely on total activity measurements can overlook promising mutants with improved expression or solubility but not necessarily higher activity.
  • Poorly soluble variants may be erroneously discarded due to undetectable activity.

Purpose of the Study:

  • To introduce split-green fluorescent protein (GFP) technology as a method to enhance protein library screening.
  • To overcome the limitations of traditional screening methods that rely solely on total activity measurements.
  • To enable the identification of improved protein variants by normalizing enzymatic activity to protein content.

Main Methods:

  • Utilized split-green fluorescent protein (GFP) technology for protein quantification.
  • Measured enzymatic activities of protein variants.
  • Normalized detected enzymatic activities by the quantified protein content using split-GFP.
  • Compared screening efficiency with and without the split-GFP normalization method.

Main Results:

  • Split-GFP technology provides a technically simple and affordable way to normalize enzymatic activity.
  • Quantification of protein content alongside activity measurement allows for a more accurate assessment of variant performance.
  • The method improves the detection of variants with enhanced expression or solubility, even if their specific activity is not increased.
  • Overall screening efficiency for identifying improved protein mutants is enhanced.

Conclusions:

  • Split-green fluorescent protein (GFP) technology significantly improves protein library screening efficiency.
  • This method allows for the detection and selection of variants with improved expression and solubility, not just activity.
  • The normalization of enzymatic activity to protein content provides a more comprehensive evaluation of protein variants for engineering purposes.